Three different classes of enzymatic activity have been shown to be required for the complete hydrolysis of cellulose to glucose. The two major activities involved in cellulose solubilization are endoglucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91) (1, 2). For the production of glucose a third type of activity, cellobiase or .beta.-glucosidase (EC 3.2.1.21) is also required. The precise manner in which these three different classes of enzyme interact to bring about the complete hydrolysis of cellulose is not yet clear.
Some filamentous fungi produce a number of different isoenzymes of each class of cellulolytic enzyme which apparently interact synergistically in hydrolysis (3, 4, 5, 6).
Trichoderma has been shown to produce at least two immunologically distinct cellobiohydrolases CBH I and CBH II, at least 2 endoglucanases, ENDO II and ENDO III, and a .beta.-glucosidase. ENDO II and ENDO III are now known as EGI and EGII, respectively. While enzymatic hydrolysis of cellulose proceeds most rapidly in the presence of all these enzymes, CBH I alone is able to degrade crystalline cellulose to glucose and cellobiose (7, 8, 9).
Two groups have reported the molecular cloning of the T. reesei gene for CBH I and the complete sequence of this gene is known (10, 11).
Yeast is an important industrial organism and is used for brewing, wine making, baking, ethanol production, single cell protein production and more recently for the production of pharmaceuticals such as interferon, growth hormone and Hepatitis B virus antigen. Yeast do not produce enzymes that degrade cellulose. The development of yeast strains able to hydrolyse cellulose would make possible improvements in existing processes where cellulose or glucans are present in the raw material used. As important would be the possibility of developing new processes not currently possible.
In filtration and clarification of beer high molecular weight .beta.-glucans originating from barley grain cause problems. In the brewing industry microbial .beta.-glucanases are used to remove these .beta.-glucans. If the yeast used in the production of beer were able to produce endoglucanases, the filterability of beer would significantly be improved and the cost of filtering would decrease. By transferring individual fungal cellulase genes to yeast it is possible to produce yeast strains that produce only one cellulase enzyme. Such yeast strains would produce enzymes for use in, for example the pulp and paper industry. Cellulose used in paper making could be swelled by pretreating with one cellulase enzyme, which would bring about swelling without excessive hydrolysis of cellulose.
There are two ways in which a foreign gene can be expressed in yeast. The simplest is to join the whole gene from the chromosome of the donor organism to a yeast vector and transform a yeast cell. If the yeast genetic system recognizes the appropriate sequences in the transferred gene the gene will be expressed. However, in practice this is rare and depends at least in part on the genetic distance between the donor organism and the yeast.
For example, of the five genes from Aspergillus niger tested in Saccharomyces cerevisiae, only one of these was found to express (12). Therefore it cannot be assumed that heterologous genes will automatically be expressed in yeast.
The second method of obtaining expression of genes in yeast is by connecting either the chromosomal gene or a cDNA copy of the messenger RNA coding for the desired gene to a yeast promotor sequence. In this way, human eukaryote interferon (13), hepatitis B virus surface antigen (14), bovine rennin (15), and mouse .alpha.-amylase (16) have all been expressed in yeast.
These and other studies show that while expression of the cDNA or gene is always obtained, the amount and cellular location of the product is very difficult to predict in the absence of experimentation. Montenecourt (1) outlined a number of possible cloning strategies for cloning cellulase genes from T. reesei but did not describe the methods to be used to achieve the goal.